Mechanisms of fusicoccin action: evidence for concerted modulations of secondary K(+) transport in a higher plant cell

Abstract

Fusicoccin (FC) has long been known to promote K(+) uptake in higher plant cells, including stomatal guard cells, yet the precise mechanism behind this enhancement remains uncertain. Membrane hyperpolarization, thought to arise from primary H(+) pumping stimulated in FC, could help drive K(+) uptake, but the extent to which FC stimulates influx and uptake frequently exceeds any reasonable estimates from Constant Field Theory based on changes in the free-running membrane potential (V m) alone; furthermore, unidirectional flux analyses have shown that in the toxin K(+) ((86)Rb(+)) exchange plummets to 10% of the control (G.M. Clint and E.A.C. MacRobbie 1984, J. Exp. Bot.35 180-192). Thus, the activities of specific pathways for K(+) movement across the membrane could be modified in FC. We have explored a role for K(+) channels in mediating these fluxes in guard cells ofVicia faba L. The correspondence between FC-induced changes in chemical ((86)Rb(+)) flux and in electrical current under voltage clamp was followed, using the K(+) channel blocker tetraethylammonium chloride (TEA) to probe tracer and charge movement through K(+)-selective channels. Parallel flux and electrical measurements were carried out when cells showed little evidence of primary pump activity, thus simplifying analyses. Under these conditions, outward-directed K(+) channel current contributed appreciably to charge balance maintainingV m, and adding 10 mM TEA to block the current depolarized (positive-going)V m; TEA also reduced(86)Rb(+) efflux by 68-80%. Following treatments with 10 μM FC, both K(+) channel current and(86)Rb(+) efflux decayed, irreversbly and without apparent lag, to 10%-15% of the controls and with equivalent half-times (approx. 4 min). Fusicoccin also enhanced(86)Rb(+) influx by 13.9-fold, but the influx proved largely insensitive to TEA. Overall, FC promotednet cation uptake in 0.1 mM K(+) (Rb(+)), despite membrane potentials which were 30-60 mVpositive of the K(+) equilibrium potential. These results tentatively link (chemical) cation efflux to charge movement through the K(+) channels. They offer evidence of an energy-coupled mechanism for K(+) uptake in guard cells. Finally, the data reaffirm early suspicions that FC alters profoundly the K(+) transport capacity of the cells, independent of any changes in membrane potential.